PROCESS FOR PURIFYING MONOCLONAL ANTIBODY

专利摘要:
The present invention relates to a method for purifying a monoclonal antibody or a fusion protein between the Fc fragment of an antibody and a second polypeptide, comprising a) a step of affinity chromatography on a resin having a matrix of cross-linked methacrylate polymer gel, to which is grafted protein A, b) a viral inactivation step, c) a cation exchange chromatography step on a resin having a reticulated agarose gel matrix, on which are grafted sulphonate groups (-SO3-) via dextran-based spacer arms; d) an anion-exchange chromatography step on a hydrophilic polyethersulfone membrane coated with a crosslinked polymer on which amine groups are grafted; quaternary (Q); and e) a nanofiltration step with a filter having an asymmetric polyethersulfone double membrane with a porosity of about 20 nm.
公开号:FR3025515A1
申请号:FR1458346
申请日:2014-09-05
公开日:2016-03-11
发明作者:Francois Coutard
申请人:LFB SA；
IPC主号:

专利说明:

[0001] FIELD OF THE INVENTION The present invention is in the field of methods of purifying monoclonal antibodies or Fc fusion proteins for pharmaceutical applications. It relates to a method for purifying a monoclonal antibody or a fusion protein between the Fc fragment of an antibody and a second polypeptide, comprising a) a step of affinity chromatography on a resin having a matrix of cross-linked methacrylate polymer, on which is grafted protein A, b) a viral inactivation step, c) a cation exchange chromatography step on a resin having a crosslinked agarose gel matrix, on which are grafted sulfonate groups (-503-) via dextran-based spacer arms; d) an anion-exchange chromatography step on a hydrophilic polyethersulfone membrane coated with a cross-linked polymer to which quaternary amine groups are grafted ( Q), and e) a nanofiltration step with a filter having a double polyethersulfone membrane having a porosity of about 20 nm. PRIOR ART During the last decade, there has been a strong development of passive immunotherapy treatments using antibodies, often monoclonal antibodies, in various therapeutic areas: cancers, prevention of alloimmunisation in pregnant women with Rhesus. negative, infectious diseases, inflammatory and especially autoimmune diseases.
[0002] In order to be used as a drug, an antibody must meet stringent requirements in terms of quality, purity, and safety. Therefore, different methods of antibody purification have been developed to meet these requirements. These methods generally involve several purification steps by chromatography, as well as one or more viral elimination or inactivation steps (Fahrner et al Biotechnol Genet Eng Rev. 2001; 18: 301-27; Liu et al. MAbs.
[0003] 2010 Sep-Oct; 2 (5): 480-99). Although many methods for obtaining purified antibodies meeting the requirements of health authorities have been described, there is nevertheless a need for optimized purification methods. Indeed, the existing processes impose on manufacturers significant production costs, which it is important to reduce as much as possible in order to reduce the cost of monoclonal antibody treatments.
[0004] The overall cost of an antibody production process varies according to a number of factors, such as in particular the cost of each of the products used for the purification, the amount of antibody that can be treated in only once, the duration of implementation of each of the process steps, and the efficiency of each of the process steps. In addition, these factors are interdependent, that is, the choice of a particular product for one of the purification steps will impact each of these factors differently. However, for each of the purification steps that can be used in a process for purifying a monoclonal antibody, the skilled person must make a choice between many commercially available products, each having its advantages and disadvantages. Thus, in the context of a purification step by chromatography, the skilled person must make choices in the chromatography resin (base and ligand) and buffers used. However, there are many chromatography resins, based on different bases (agarose, dextran, synthetic polymers, etc.) and different ligands (affinity, cation exchange, ion exchange, hydrophobic interactions, etc.). .), and several buffers are likely to be used for each chromatography resin. It is therefore particularly difficult for a person skilled in the art to select a combination of steps and specific products capable of significantly reducing the purification costs of a monoclonal antibody, while maintaining the level of quality, purity, and safety of the purified antibody. SUMMARY OF THE INVENTION In the context of the present invention, the inventors have demonstrated that a particular combination of purification and viral elimination or inactivation steps makes it possible to reduce by a factor of 3 the costs of purification of a monoclonal antibody, while maintaining the level of quality, purity, and safety of the purified antibody. This sharp decrease in the cost of purification of a monoclonal antibody has been made possible by the selection of less expensive products, making it possible to treat a higher amount of antibody at one time, and by optimizing the operating conditions of each step. to maintain the level of quality, purity, and safety of the purified antibody. In a first aspect, the present invention thus relates to a method of purifying a monoclonal antibody or a fusion protein between the Fe fragment of an antibody and a second polypeptide, comprising: a) a chromatography step of affinity to a resin having as a matrix a crosslinked methacrylate polymer gel, onto which is grafted protein A, b) a viral inactivation step, c) a cation exchange chromatography step on a resin having a matrix a crosslinked agarose gel, to which sulfonate groups (-503-) are grafted via dextran-based spacer arms; d) an anion-exchange chromatography step on a hydrophilic polyethersulfone membrane coated with a crosslinked polymer on which are grafted quaternary amine groups (Q), and e) a nanofiltration step with a filter having a double polyethersulfone membrane with a porosity of about 20 nm.
[0005] Advantageously, the crosslinked methacrylate polymer gel on which is grafted protein A used in step a) is in the form of beads having a mean diameter of between 30 and 60 [Inn, preferably between 40 and 50 [Inn. In addition, the elution buffer used in step a) to elute the antibody is preferably a formate buffer, which is advantageously used at a molarity of 5 to 10 mM and a pH of between 2, 6 and 3.6. Advantageously, step b) is carried out by incubation for 30 to 120 minutes at a temperature of 20 to 25 ° C. in a medium comprising 0.5 to 2% (v / v) of polyoxyethylene-p-octylphenol (Triton X- 100, CAS No. 9002-93-1). Advantageously, the buffer used in step d) is a trishydroxymethylaminomethane (TRIS) buffer at a concentration of 15 to 25 mM, a pH of 7.5 to 8.5 and a conductivity of 5 to 15 mS / cm. Advantageously, step e) further comprises prior filtration through a VPF pre-filter (Viresolve PreFilter, depth filter comprising cellulose fibers, diatomaceous earth and a negatively charged resin) or Viresolve pro Shield ( polyethersulfone membrane with a porosity of 0.22 [Inn functionalized with 503- groups). Advantageously, the method further comprises a step of ultrafiltration and / or diafiltration. Advantageously, the method according to the invention is carried out on a culture supernatant of a clone producing the monoclonal antibody or the fusion protein between the Fe fragment of an antibody and a second polypeptide. Advantageously, the method according to the invention is used for the purification of a monoclonal antibody, in particular of an antibody directed against one of the following antigens: Rhesus D, CD2, CD3, CD4, CD19, CD20, CD22 , CD25, CD30, CD33, CD40, CD51 (Integrin alpha-V), CD52, CD80, CTLA-4 (CD152), SLAMF7 (CD319), Her2 / neu, EGFR, EPCAM, CCR4, CEA, FR-alpha, GD2, GD3, HLA-DR, IGF1R (CD221), phosphatidylserine, TRAIL-R1, TRAIL-R2, Clostridium difficile antigens, Staphylococcus aureus antigens (in particular CIfA and lipothric acid), cytomegalovirus antigens (in particular glycoprotein B), antigens of Escherichia coli (in particular Shiga-like toxin, subunit IIB), respiratory syncytial virus antigens (Protein F in particular), antigens of the hepatitis B virus, antigens of the influenza virus 3025515 4 A (haemagglutinin in particular), antigens of Pseudomonas aeruginosa serotype IATS 011, antigens of rabies virus (Glycoprotein in particular), o Phosphatidylserine. DESCRIPTION OF THE FIGURES FIG. 1. Determination of the injected volume at the 10% BT point for purification by Protein A affinity chromatography on MabSelect SuReTM (A), Poros GoPureTM (B), Toyopearl AF-rProtein A- 650F (C) and AmsphereTM Protein A JWT203 (D) Figure 2. Analysis by Design-Experte Software of the Neutralized Eluate Disorder (Just After Neutralization, A to D) and Stabilized Neutralized Eluates (1 hour after neutralization, E at H) following elution of the protein A column by a maleate buffer (A and E), acetate (B and F), formate (C and G) or citrate (D and H), depending on the pH (represented on the abscissa) and the molarity (represented on the ordinate) of the buffer. For a given pair (pH / molarity), the haze of each eluate is denoted by a value varying between 0 and 3, a value of 0 corresponding to a clear eluate and a value of 3 to a highly disturbed eluate (opalescent). The curves representing the pairs (pH / molarity) corresponding to a given disorder value are represented. Figure 3. Analysis of the percentage of monomers in neutralized eluates following elution of the protein A column by a maleate (A), acetate (B), formate (C) or citrate (D) buffer, as a function of pH ( represented on the abscissa) and the molarity 20 (represented on the ordinate) of the buffer. The curves representing the pairs (pH / molarity) corresponding to a percentage value of monomeric forms of the antibody in the neutralized eluate are shown. Figure 4. Filtration rate (g / h / m2) versus antibody load (g / m2) for nanofiltration on a Planova® 15N filter (A), Planova® 20N (B), or Viresolve® Pro 20N (C). Figure 5. Antibody load (g / m2) nanofiltered versus filtration time (minutes) for nanofiltration on a Planova® 15N, Planova® 20N, or Viresolve® Pro 20N filter. DETAILED DESCRIPTION OF THE INVENTION As previously indicated, monoclonal antibody purification methods impose significant production costs on manufacturers, which are important to minimize to the extent of reducing the cost of monoclonal antibody treatments. However, the existence of many distinct commercial products which can be used for the purification of monoclonal antibodies and the very great possibility of variations in the operating conditions of each step makes the selection of a combination of steps, products and appropriate operating conditions to reduce the overall purification cost extremely difficult for those skilled in the art.
[0006] However, the inventors have now demonstrated that a particular combination of purification and viral elimination or inactivation steps makes it possible to reduce by a factor of 3 the costs of purification of a monoclonal antibody, while maintaining the level of quality, purity, and safety of the purified antibody. This sharp decrease in the cost of purification of a monoclonal antibody has been made possible by the selection of less expensive products, products making it possible to treat a higher amount of antibody at one time, and by optimizing the operating conditions of each step in order to maintain the level of quality, purity, and safety of the purified antibody.
[0007] The present invention thus relates to a method of purifying a monoclonal antibody or a fusion protein between the Fc fragment of an antibody and a second polypeptide, comprising: a) a step of affinity chromatography on a resin having for matrix a crosslinked methacrylate polymer gel, on which is grafted protein A, b) a viral inactivation step, c) a cation exchange chromatography step on a resin matrix crosslinked agarose gel on which sulfonate groups (-503-) are grafted via dextran spacer arms; d) an anion exchange chromatography step on a hydrophilic polyethersulfone membrane coated with a cross-linked polymer on which are grafted quaternary amine groups (Q), and e) a nanofiltration step with a filter having a double polyethersulfone membrane with a porosity of about 20 nm.
[0008] Starting material The method according to the invention is applicable both to the purification of a monoclonal antibody and of a fusion protein between the Fc fragment of an antibody and a second polypeptide. Indeed, the first step (step a)) is a step of affinity chromatography on a resin carrying protein A, a Staphylococcus aureus protein which specifically binds to the Fc fragment of the antibodies, and in particular to the human Fc fragment. . By "antibody" or "immunoglobulin" antibody is meant a molecule comprising at least one antigen binding domain and a constant domain comprising a Fc fragment capable of binding to FcR receptors. In most mammals, such as humans and mice, an antibody is composed of 4 polypeptide chains: 2 heavy chains and 2 light chains linked together by a variable number of disulfide bridges providing flexibility to the molecule. Each light chain consists of a constant domain (CL) and a variable domain (VL); the heavy chains being composed of a variable domain (VH) and 3 or 4 constant domains (CH1 to CH3 or CH1 to CH4) depending on the isotype of the antibody. In a few rare mammals, such as camels and llamas, the antibodies consist of only two heavy chains, each heavy chain comprising a variable domain (VH) and a constant region. Variable domains are involved in antigen recognition, while constant domains are involved in the biological, pharmacokinetic and effector properties of the antibody. Unlike variable domains whose sequence varies strongly from one antibody to another, the constant domains are characterized by an amino acid sequence very close to one antibody to another, characteristic of the species and the isotype. , with possibly some somatic mutations. The Fc fragment is naturally composed of the constant region of the heavy chain excluding the CH1 domain, i.e., the lower hinge region and the constant CH2 and CH3 or CH2 to CH4 domains (according to US Pat. isotype). In human IgG1, the complete Fc fragment is composed of the C-terminal portion of the heavy chain from the cysteine residue at position 226 (C226), the numbering of amino acid residues in the Fc fragment being throughout the present invention. description that of the EU index described in Edelman et al (Edelman, GM et al., Proc Natl Acads USA, 63, 78-85 (1969)) and Kabat et al (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The corresponding Fc fragments of other types of immunoglobulins can be easily identified by those skilled in the art by sequence alignments. The Fcγ fragment is glycosylated at the CH2 domain with the presence, on each of the 2 heavy chains, of an N-glycan linked to the asparagine residue at position 297 (Asn 297). The following binding domains, located in Fcγ, are important for the biological properties of the antibody: FcRn receptor binding domain, involved in the pharmacokinetic properties (in vivo half-life) of the antibody: Different data suggest that some residues at the CH2 and CH3 domain interface are involved in FcRn receptor binding. C1q complement protein binding domain, involved in the CDC response (for "complement dependent cytotoxicity"): located in the CH2 domain; - FcR receptor binding domain, involved in phagocytosis or ADCC type responses (for "antibody-dependent cellular cytotoxicity): located in the CH2 domain. Within the meaning of the invention, the Fc fragment of an antibody may be natural, as defined above, or may have been modified in various ways, provided that it is capable of binding protein A. Such modifications may include the deletion of certain portions of the Fc fragment, provided that the Fc fragment thus obtained is capable of binding protein A. The modifications may also include different amino acid substitutions that may affect the biological properties of the protein. the antibody, provided that the Fc fragment thus obtained is capable of binding to protein A. In particular, when the antibody is an IgG, it may comprise mutations intended to increase the binding to the FcγRIII receptor (CD16), as disclosed in W000 / 42072, Shields et al., 2001, Lazar et al (Lazar, GA, et al., Proc Natl Acad Sci US A 103 (11): 4005-10), WO2004 / 029207, WO / 2004063351, W02004 / 074455. Mutations to increase FcRn receptor binding and thus in vivo half-life may also be present as described, for example, in Shields et al (Shields RL, et al., J Biol Chem.
[0009] 2001 Mar 2; 276 (9): 6591-604), Dall'Acqua et al. 2002, Hinton et al. 2004, Dall'Acqua et al. 2006 (a), W000 / 42072, W002 / 060919A2, WO2010 / 045193 , or W02010 / 106180A2. Other mutations, such as those for decreasing or increasing complement protein binding and therefore CDC response, may or may not be present (see WO99 / 51642, WO2004074455A2, EE Idusogie et al, J Immunol 2001; 166: 2571-5, Dall'Acqua et al., J Immunol 2006; 177: 1129-1138, and Moore GL et al., MAbs 2: 2, 181189; March / April, 2010). By "monoclonal antibody" or "monoclonal antibody composition" is meant a composition comprising antibody molecules having identical and unique antigenic specificity. The antibody molecules present in the composition are likely to vary in their posttranslational modifications, and in particular in their glycosylation structures or their isoelectric point, but have all been coded by the same heavy and light chain sequences and therefore, before any post-translational modification, the same protein sequence. Certain protein sequence differences, related to post-translational modifications (eg cleavage of C-terminal heavy chain lysine, deamidation of asparagine residues and / or isomerization of aspartate residues), may nevertheless exist between the different antibody molecules present in the composition.
[0010] The purified monoclonal antibody within the scope of the invention may advantageously be chimeric, humanized, or human. Indeed, the protein A of Staphylococcus aureus has a particular affinity for binding to human Fc fragments, and in particular to the human Fcγ fragment. By "chimeric antibody" is meant an antibody which contains a naturally occurring variable (light chain and heavy chain) region derived from an antibody of a given species in association with the constant light chain and heavy chain regions of a antibody of a heterologous species to said given species. Advantageously, if the monoclonal antibody composition for use as a drug according to the invention comprises a chimeric monoclonal antibody, it comprises human constant regions. Starting from a non-human antibody, a chimeric antibody can be prepared using genetic recombination techniques well known to those skilled in the art. For example, the chimeric antibody can be made by cloning for the heavy chain and the light chain a recombinant DNA comprising a promoter and a sequence coding for the variable region of the non-human antibody, and a sequence coding for the constant region. a human antibody.
[0011] For methods of preparing chimeric antibodies, for example, reference may be made to Verhoeyn et al (Verhoeyn et al., BioEssays, 8:74, 1988). The term "antibody-humanized" is intended to mean an antibody which contains CDRs regions derived from an antibody of non-human origin, the other parts of the antibody molecule being derived from one (or more) human antibodies. In addition, some of the skeletal segment residues (referred to as FR) may be modified to maintain binding affinity (Jones et al., Nature, 321: 522-525, 1986, Verhoeyen et al., 1988, Riechmann et al. Nature, 332: 323-327, 1988). The humanized antibodies according to the invention can be prepared by techniques known to those skilled in the art such as CDR grafting, resurfacing, superhumanization, and string content technologies. libraries ", de - Guided selection", de - FR shuffling "and" Humaneering ", as summarized in the review by Almagro et al (Almagro et al., Frontiers in Bioscience 13, 1619-1633, January 1, 2008). By "human antibody" is meant an antibody whose entire sequence is of human origin, i.e. whose coding sequences have been produced by recombination of human genes encoding the antibodies. Indeed, it is now possible to produce transgenic animals (eg mice) that are capable, upon immunization, of producing a complete repertoire of human antibodies in the absence of endogenous production of immunoglobulin (see Jakobovits et al. Proc Natl Acad Sci USA 90: 2551 (1993), Jakobovits et al., Nature 362: 255-258 (1993), Bruggermann et al., Year in Immuno., 7:33 ( 1993), Duchosal et al., Nature 355: 258 (1992), U55,591,669, US 5,598,369, US 5,545,806, US 5,545,807 and US 6,150,584. Human antibodies can also be obtained from phage display libraries (Hoogenboom et al., J. Mol Biol., 227: 381 (1991), Marks et al., J. Mol Biol., 222: 581). 5,597 (1991), Vaughan et al Nature Biotech 14: 309 (1996)).
[0012] The antibodies may be several isotypes, depending on the nature of their constant region: the constant regions y, a, p, e and δ correspond respectively to the immunoglobulins IgG, IgA, IgM, IgE and IgD. Advantageously, the monoclonal antibody present in a composition used as a medicament in the context of the invention is of IgG isotype. Indeed, this isotype shows an ability to generate ADCC ("Antibody-Dependent Cellular Cytotoxicity", or antibody-dependent cellular cytotoxicity) activity in the largest number of (human) individuals, and is therefore primarily used for pharmaceutical applications of monoclonal antibodies. In addition, protein A has a particular affinity for binding to the human Fcy fragment.
[0013] The constant regions include several subtypes: y1, y2, y3, these three types of constant regions having the particularity of binding human complement, and y4, thereby creating the IgG1, IgG2, IgG3, and IgG4. Advantageously, the monoclonal antibody present in a composition used as a medicament in the context of the invention is of IgG1 isotype. Indeed, the Fcγ1 fragment has a particularly important binding affinity for protein A.
[0014] The monoclonal antibody composition to be purified by the method according to the invention can be produced by a cell clone, a transgenic non-human animal or a transgenic plant, by technologies well known to those skilled in the art.
[0015] In particular, cellular clones producing the antibody composition to be purified can be obtained by 3 main technologies: 1) Obtaining a hybridoma by fusion of a B lymphocyte producing the antibody of interest with an immortalized line, 2) Immortalization of a B lymphocyte producing the antibody of interest by the Epstein-Barr virus (EBV), 3) Isolation of the sequences coding for an antibody of interest (generally from a hybridoma or a B lymphocyte) immortalized), cloning into one or more expression vector (s) of the sequences coding for the heavy and light chains of the antibody, transformation of a cell line by the expression vector (s), and separation of the different cellular clones obtained. An expression vector of the heavy and light chains of the antibody comprises the elements necessary for the expression of the sequences coding for the heavy and light chains of the antibody, and in particular a promoter, a codon for initiation of the transcription, termination sequences, and appropriate transcriptional regulatory sequences. These elements vary according to the host serving for the expression and are readily selected by those skilled in the art in view of his general knowledge. The vector may especially be plasmidic or viral. Transformation techniques are also well known to those skilled in the art.
[0016] The transformation of cell lines by one or more expression vectors of the sequences coding for the heavy and light chains of the antibody is the most commonly used, in particular for obtaining chimeric or humanized antibodies. The transformed cell line is preferably of eukaryotic origin, and may especially be selected from insect, plant, yeast or mammalian cells. The antibody composition can then be produced by culturing the host cell under appropriate conditions. Cell lines suitable for the production of antibodies include the lines selected from: SP2 / 0; YB2 / 0; IR983F; human myeloma Narnalwa; PERC6; the CHO lines, in particular CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr-, or CHO line deleted for the two alleles coding for the FUT8 gene and / or the GMD gene; Wil-2; Jurkat; Vero; Molt 3025515 COS-7; 293-HEK; BHK; K6H6; NSO; SP2 / 0-Ag 14, P3X63Ag8.653, embryonic duck cell line EB66® (Vivalis); and H4-II-E rat hepatoma lines (DSM ACC3129), H4-II-Es (DSM ACC3130) (see WO2012 / 041768) In a preferred embodiment, the antibody is produced in one of the lines following: YB2 / 0; CHO line 5 deleted for the two alleles coding for the FUT8 gene and / or the GMD gene; embryonic duck cell line EB66® (Vivalis); and H4-II-E rat hepatoma lines (DSM ACC3129), H4-II-Es (DSM ACC3130). In a preferred embodiment, the antibody is produced in YB2 / 0 (ATCC CRL-1662).
[0017] Alternatively, the antibody composition to be purified can be produced in a transgenic non-human animal. A transgenic non-human animal can be obtained by direct injection of the gene (s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) into a fertilized egg (Gordon et al. ). A non-human transgenic animal can also be obtained by introducing the gene (s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) into an embryonic stem cell and preparing the gene. by a chimera aggregation method or a chimeric injection method (see Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994), Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993)). A transgenic non-human animal can also be obtained by a cloning technique in which a nucleus, in which the gene (s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) has been introduced, is transplanted into an enucleated egg (Ryan et al., 1997 Science, 278: 873-8756, Cibelli et al., 1998 Science, 280: 1256-1258, W00026357A2). A transgenic non-human animal producing an antibody of interest can be prepared by the above methods. The antibody can then be accumulated in the transgenic animal and harvested, in particular from the milk or eggs of the animal. For the production of antibodies in the milk of transgenic nonhuman animals, methods of preparation are described in particular in WO9004036A1, WO9517085A1, WO00126455A1, WO2004050847A2, WO2005033281A2, WO2007048077A2. Methods of purifying proteins of interest from milk are also known (see W00126455A1, WO2007106078A2). Non-human transgenic animals of interest include mice, rabbits, rats, goats, cattle (especially cows), and poultry (especially chicken). The antibody composition to be purified can also be produced in a transgenic plant. Many antibodies have already been produced in transgenic plants and the technologies necessary to obtain a transgenic plant expressing an antibody of interest and to the recovery of the antibody are well known to those skilled in the art (see Stoger E, et al Molecular Breeding 9: 149-158, 2002, Fisher 302551511 R, et al., Vaccine 21 (2003) 820-825, Ma JK, et al., Nat Rev Genet.
[0018] 2003 Oct; 4 (10): 794-805; Schillberg S, et al. Vaccine 23 (2005) 1764-1769). It is also possible to influence the glycosylation obtained in the plants to obtain glycosylation close to that of natural human antibodies (without xylose), but with, in addition, low fucosylation, for example using small interfering RNAs (Forthal et al., J Immunol 2010; 185; 6876-6882). The monoclonal antibody to be purified can be directed against any antigen of interest, and in particular against the following antigens: - Rhesus D, the anti-Rhesus D antibodies being useful for the prevention of alloimmunization in Rh-negative individuals, - Antigens expressed by cancer cells, which may be targeted in the treatment of cancers, and in particular: CD20, Her2 / neu, CD52, EGFR, EPCAM, CCR4, CTLA-4 (CD152), CD19, CD22, CD3, CD30 , CD33, CD4, CD40, CD51 (Integrin alpha-V), CD80, CEA, FR-alpha, GD2, GD3, HLA-DR, IGF1R (CD221), phosphatidylserine, SLAMF7 (CD319), TRAIL-R1, TRAIL- R2. Antigens expressed by cells infected with pathogens, capable of being targeted in the treatment of infections by pathogens, and in particular: antigens of Clostridium difficile, antigens of Staphylococcus aureus (in particular CIfA and lipothric acid), antigens cytomegalovirus (including glycoprotein B), Escherichia coli antigens (including Shiga-like toxin, subunit IIB), respiratory syncytial virus antigens (Protein F in particular), hepatitis B virus antigens, influenza virus antigens A (Hemagglutinin in particular), Pseudomonas aeruginosa serotype IATS 011 antigens, rabies virus antigens (glycoprotein in particular), phosphatidylserine. Antigens expressed by immune cells, which may be targeted for the treatment of autoimmune diseases, and in particular: CD20, CD52, CD25, CD2, CD22, CD3, and CD4.
[0019] Fc fusion protein - fusion protein between the Fc fragment of an antibody and a second polypeptide "or Fc fusion protein" means a protein comprising an antibody Fc fragment operably linked to a second polypeptide . Such an Fc fusion protein comprises an Fc fragment conferring on it the effector and pharmacological properties of an antibody (as well as the ability to bind to protein A), and a second polypeptide (fusion partner) conferring other biological properties. Fc fusion proteins, as well as monoclonal antibodies, comprise an Fc fragment binding to protein A. Therefore, the technical teachings obtained by the inventors on monoclonal antibodies apply directly to Fc fusion proteins.
[0020] The second polypeptide or fusion partner can in particular be chosen from a receptor (or the binding domain of a receptor to its ligand), a ligand (or the binding domain of a ligand to its receptor), a molecule adhesion, a cytokine, a chemokine, or any other protein or domain of a protein.
[0021] The fusion between the Fc fragment and the second polypeptide may be direct or indirect via a linker, which may in particular consist of one or more amino acids of the glycine or serine type. Such Fc fusion proteins have been developed for different therapeutic applications. In particular, the following Fc fusion proteins are capable of being purified using the method according to the invention: abatacept and belatacept: fusion proteins between the CTLA-4 ectodomain and the Fc of an IgG1, used in immunosuppression in rheumatoid arthritis (abatacept) and transplantation (belatacept) - etanercept: fusion protein between TNF-RII receptor ectodomain and Fc of IgG1, used in the treatment of rheumatoid arthritis and psoriasis, - alefacept: fusion protein between LFA-3 ectodomain (CD58) and Fc of IgG1, used therapeutically in the treatment of psoriasis, or - rilonacept: dimeric fusion protein consisting of binding domains extracellular portions of interleukin-1 type I receptor (IL-1RI) and an IL-1 receptor accessory protein (IL-1RAcP) linearly bound to the Fc portion of human immunoglobulin IgG1, used in the t and severe forms of Cryopyrin Associated Periodic Syndromes (CAPS), including Familial Autoinflammatory Cold Syndrome (FCAS) and Muckle-Wells Syndrome (MWS). atacicept: a recombinant fusion that contains the soluble TACI receptor associated with the Fc fragment of a human IgG1, used in the treatment of rheumatoid arthritis, systemic lupus and multiple sclerosis. briobacept: fusion protein consisting of the BAFF receptor and a constant fragment of IgG1, used in the treatment of rheumatoid arthritis. Such Fc fusion proteins are produced recombinantly by any appropriate technology chosen in particular from those described above for the production of recombinant monoclonal antibodies (transformed cell clone, transgenic non-human animal, transgenic plant in particular).
[0022] Product to be purified The purification process according to the invention is advantageously carried out starting from a composition comprising the antibody in unpurified form, that is to say also comprising other contaminating products (other proteins, DNA , sugars, etc ...).
[0023] The method according to the invention can thus be used in particular using the following materials: - culture supernatant of a clone producing the monoclonal antibody or the fusion protein between the Fc fragment of an antibody and a second polypeptide, - milk of a transgenic non-human animal expressing the monoclonal antibody or the fusion protein between the Fc fragment of an antibody and a second polypeptide, or - cell extract of a transgenic plant expressing the monoclonal antibody or the fusion protein between the Fc fragment of an antibody and a second polypeptide. Many monoclonal antibodies or fusion proteins between the Fc fragment of an antibody and a second polypeptide are produced in transformed cell clones, and the method according to the invention can therefore advantageously be carried out on a culture supernatant. a clone producing the monoclonal antibody or the fusion protein between the Fc fragment of an antibody and a second polypeptide. A clone producing the monoclonal antibody or the fusion protein between the Fc fragment of an antibody and a second polypeptide is understood to mean a transformed cell (which may be chosen in particular from those described above) by a vector. expressing the monoclonal antibody or the fusion protein between the Fc fragment of an antibody and a second polypeptide. By "culture supernatant" is meant the composition obtained by centrifugation of the culture medium of the cells of the producing clone and exclusion of the cells or debris present in the culture medium, said culture medium possibly optionally having been subjected beforehand to a step lysis of the cells of the producing clone. Step a) Step a) of the process according to the invention is a step of affinity chromatography on a resin having as a matrix a crosslinked methacrylate polymer gel, on which is grafted protein A. This step makes it possible to purify very importantly the Fc antibody or fusion protein, because of the high affinity and high specificity of protein A for the Fc fragment of the antibodies. Protein A Affinity chromatography specifically separates molecules that bind to a particular ligand. In the context of the purification of antibodies, a protein A affinity chromatography step is commonly used, this protein binding specifically to the Fc fragment of the antibodies, in particular to the human Fc fragment, and more particularly to the Fcγ fragment. and especially human Fey1.
[0024] "Protein A" means the Staphylococcus aureus protein encoded by the Spa gene, or a derivative or a fragment of this protein capable of binding to the Fc fragment of a monoclonal antibody. The Staphylococcus aureus protein A encoded by the spa gene is a membrane protein of Staphylococcus aureus comprising N-terminal 5 homologous domains (EDABC) each capable of binding to the Fc fragment of the antibodies, the C-terminal region (X) serving to anchor the protein in the bacterial membrane. Native protein A can be isolated directly from protein A secreting Staphylococcus aureus cultures, or recombinant Escherichia coli (E. coli) bacteria expressing protein A (Ldfdahl et al., Proc Natl Acad Sci U S A.
[0025] 1983 Feb; 80 (3): 697-701; Uhlén et al. J Biol Chem.
[0026] 1984 Feb 10; 259 (3): 1695-702). In order to optimize its use for affinity chromatography, various fragments or variants of proteins A capable of binding specifically and with high affinity to the Fc fragment of the antibodies have been proposed. In particular, different domains of the native protein A or derivatives and / or fragments of these domains have been proposed in repeated form (especially dimers, tetramers or hexamers) for the purification of antibodies. Thus, a "B" fragment of the B domain of protein A has been proposed for use in the purification of antibodies (US6,013,763). Different variants of recombinant protein A or recombinant protein fragment comprising a cysteine residue or an N terminal arginine residue allowing easier attachment to the chromatography matrix have also been described (US5,084,559; US5,260,373; 399.750). Protein A variants or functional fragments of protein A having improved stability under alkaline conditions have also been described (US7, 709, 209, WO2012 / 083425), these variants being useful to allow repeated sanitization of the affinity chromatography columns at the same time. Protein A without excessive release of protein A. Affinity chromatography matrix In the method according to the invention, protein A is attached to an affinity chromatography matrix consisting of a crosslinked methacrylate polymer gel. By "crosslinked methacrylate polymer" is meant any crosslinked polymer or co-polymer comprising methacrylate monomers. "Methacrylate" means the methacrylate ion of formula (CH2 = C (CH3) C00-), as well as the salts and esters of this ion. Indeed, the inventors have demonstrated that this particular type of resin makes it possible to increase the purified antibody load in one go, to guarantee a good yield (at least 90%) and to obtain an eluate at the same time. clear appearance (see Example 1). The crosslinked methacrylate polymer gel on which is grafted protein A used in step a) may advantageously be in the form of beads having a mean diameter of between 30 and 60 [Inn, advantageously between 40 and 50 [Inn, and in particular about 45, 49 or 50 [Inn.
[0027] Depending on the protein A used, it may be attached to the affinity chromatography matrix by various customary coupling types, such as CNBr multipoint coupling (coupling between primary amino functions of protein A and an activated matrix). by CNBr), the single-point coupling by a thioether bond between the matrix and a cysteine residue of protein A, obtained in particular by activation of the matrix with an epoxide or an epichlorohydrin. Examples of matrices consisting of a cross-linked methacrylate polymer gel in the form of beads having a mean diameter of between 40 and 50 [Inn, on which is grafted protein A, include the following matrices: TOYOPEARL® AF-rProtein A HC-650F (hydroxylated methacrylate polymer matrix in the form of beads with an average diameter of 45 [Inn, on which is grafted recombinant protein A, marketed by TOSOH BIOSCIENCE), Annsphere 'Protein A JWT203 (polymer resin) methacrylate in bead form with an average diameter of 49 [Inn, on which is grafted an alkaline stable modified domain C tetramer produced in E. coli, marketed by JSR Corporation). Advantageously, the matrix resin having a crosslinked methacrylate polymer gel on which protein A is grafted is chosen from a matrix of hydroxylated methacrylate polymer in the form of beads with an average diameter of 45 [Inn, on which is grafted recombinant protein A (in particular TOYOPEARL® AF-rProtein A resin HC-650F) and a methacrylate polymer resin in the form of beads with an average diameter of 49 [Inn, on which is grafted a domain tetramer C modified alkali-stable produced in E. coli (especially AmsphereTM resin Protein A JWT203), more preferably the resin is a methacrylate polymer resin in the form of beads with an average diameter of 49 [Inn, on which is grafted an alkaline-stable modified domain C tetramer produced in E. coli (especially AmsphereTM Protein A resin JWT203). Elution Buffer In step a), the monoclonal antibody or Fc fusion protein composition to be purified is injected onto a resin having a cross-linked methacrylate polymer gel matrix on which is grafted with the protein. A, equilibrated with a neutral pH buffer, to which the monoclonal antibody or Fc fusion protein will bind via the Fc fragment. The resin is then washed to remove contaminants not bound to the resin, and then the monoclonal antibody or Fc fusion protein is eluted with a buffer to break the binding between protein A and Fc fragment. antibody or Fc fusion protein. Different types of buffers can be used for elution. In particular, elution can be achieved at acidic pH, and buffers using different weak acids can therefore be used for elution.
[0028] However, in the context of the present invention, the inventors have demonstrated that it is particularly advantageous to use a sodium formate buffer. Advantageously, the sodium formate buffer is used at a molarity of 5 to 10 mM, advantageously 5 to 9 nnM, 5 to 8 nnM, 5 to 7 nnM, 5 to 6 nnM, 6 to 10 5 nM, from 6 to 9 nM, from 6 to 8 nM, from 6 to 7 nM, from 7 to 10 nnM, from 7 to 9 nnM, from 7 to 8 nnM, from 8 to 10 mM, from 8 to 9 mM, or from 9 to 10 mM, more preferably 5 to 9 mM, 5 to 8 mM, 5 to 7 mM, 5 to 6 mM and especially about 5 mM. Advantageously, the sodium formate buffer is used at a pH of between 2.6 and 3.6, advantageously between 2.7 and 3.6, between 2.8 and 3.6, and between 2.9 and 3.6. between 3.0 and 3.6, between 3.1 and 3.6, between 3.2 and 3.6, between 3.3 and 3.6, between 3.4 and 3.6, between 3, 5 and 3.6, between 2.6 and 3.5, between 2.7 and 3.5, between 2.8 and 3.5, between 2.9 and 3.5, 3.5, between 3.1 and 3.5 and 3.5, between 3.2 and 3.5, between 3.3 and 3.5, between 3.4 and 3.5, 3.4, between 2.7 and 3.4, between 2.8 and 3.5, 3.4, between 2.9 and 3.4, between 3.0 and 3.4, 3.4, between 3.2 and 3.4, between 3.3 and 3.4, between 2.6 and 3 , 3, between 2.7 and 3.3, 3.3, between 2.9 and 3.3, between 3.0 and 3.3, between 3.1 and 3.3, between 3.2 and 3 , 3, 3.2, between 2.7 and 3.2, between 2.8 and 3.2, between 2.9 and 3.2, between 3.0 and 3.2, 3.2, between 2, 6 and 3.1, between 2.7 and 3.1, between 2.8 and 3.1, between 2.9 and 3.1, 3.1, between 2.6 and 3.0, between 2.7 and 3.1, and 3.0, between 2.8 and 3.0, between 2.9 and 3.0, between 3.0 and 2.6 and between 3.1 and 2.8 and between 2.6 and between 3 and 3.0 , 1 and between 3.0 and 2.6 and 2.9, between 2.7 and 2.9, between 2.8 and 2.9, between 2.6 and 2.8, between 2.7 and 2.8, or between 2.6 and 2.7 more preferably between 2.7 and 3.5, between 2.8 and 3.4, between 2.9 and 3.3, between 3.0 and 3.2, and even about 3.1. Advantageously, the sodium formate buffer is used: at a molarity of 5 to 10 mM, advantageously 5 to 9 mM, 5 to 8 mM, 5 to 7 nnM, 5 to 6 nnM, 6 to 10 nnM from 6 to 9 nnM, from 6 to 8 nnM, from 6 to 7 nnM, from 25 to 10 nnM, from 7 to 9 nnM, from 7 to 8 nnM, from 8 to 10 nnM, from 8 to 9 nnM, or from 9 to 10 mM, more preferably from 5 to 9 nnM, from 5 to 8 nnM, from 5 to 7 nnM, from 5 to 6 nnM and especially about 5 mM, and - at a pH between 2.6 and 3.6, 2.6 and 3.6, preferably between 2.7 and 3.6, between 2.8 and 3.6, between 2.9 and 3.6, between 3.0 and 3.6 between 3.1 and 3.6, between 3.2 and 3.6, between 3.3 and 3.6, between 3.4 and 3.6, between 3.5 and 3.6, between 6 and 3.5, between 2.7 and 3.5, between 2.8 and 3.5, between 2.9 and 3.5, between 3.0 and 3.5, between 3.1 and 3.5 between 3.2 and 3.5, between 3.3 and 3.5, between 3.4 and 3.5, between 2.6 and 3.4, between 2.7 and 3.4, between 2.8 and 3.5, and 3.4, between 2.9 and 3.4, between 3.0 and 3.4, between 3.1 and 3.4, between 3.2 and 3.4, between 3.3 and 3.4, between 2.6 and 3.3 between 2.7 and 3.3, between 2.8 and 3.3, between 2.9 and 3.3, between 3.0 and 3.3, between 3.1 and 3.3, between 3, 2 and 3.3, between 2.6 and 3.2, between 2.7 and 3.2, between 2.8 and 3.2, between 2.9 and 3.2, between 3.0 and 3.2 , between 3.1 and 3.2, between 2.6 and 3.1, between 2.7 and 3.1, between 2.8 and 3.1, between 2.9 and 3.1, between 3.0 and 3.1, between 2.6 and 3.0, between 2.7 and 3.0, between 2.8 and 3.0, between 2.9 and 3.0, between 2.6 and 2.9, between 2.7 and 2.9, 40 between 2.8 and 2.9, between 2.6 and 2.8, between 2.7 and 2.8, or between 2.6 and 2.7, plus 3025515 17 advantageously between 2.7 and 3.5, between 2.8 and 3.4, between 2.9 and 3.3, between 3.0 and 3.2, or even about 3.1. In fact, this type of buffer makes it possible to obtain a satisfactory yield, a clear or slightly opalescent eluate with a satisfactory volume, as well as a very small proportion of antibody aggregates. This is not the case for buffers such as sodium acetate dihydrate or trisodium citrate dihydrate buffers, which lead to moderately or strongly opalescent eluates, or the maleic acid buffer, 0.5M NaOH which leads to significant aggregate formation. of antibodies (see Example 1). After the elution of the antibody or fusion protein Fe, the eluate can be neutralized, that is to say brought to a pH of between 5 and 7, in particular between 5.5 and 6.5, and in particular about 6.0. This neutralization can in particular be carried out by adding an appropriate amount of 1M Tris buffer pH 7.5 or 1M sodium hydroxide (NaOH).
[0029] Thus, the specific choices made by the inventors concerning the affinity chromatography matrix and the elution buffer make it possible to significantly reduce the cost of this step (increased charge, very good yield, relatively cheap matrix), while ensuring a strong purification and a good quality of the purified antibody (clear or weakly opalescent eluate, very low proportion of aggregates). Step b) Step b) of the process according to the invention is a viral inactivation step. By "viral inactivation step" is meant a step in which viruses are not removed from the solution (antigens may still be detected), but are rendered inactive and therefore harmless. These steps include dry heating, pasteurization, and solvent-detergent or detergent-only treatment. These different viral inactivation steps are well known to those skilled in the art (see, in particular, the WHO guidelines for inactivation and viral elimination procedures for ensuring viral safety of human plasma derivatives). available on the WHO website). Advantageously, in the method according to the invention, the viral inactivation step is a solvent-detergent treatment or detergent treatment alone step. A solvent-detergent treatment is carried out by treating the solution with a solvent mixture, especially tri- (N-butyl) -phosphate (TnBP), and a detergent, in particular Polysorbate 80 (Polyoxyethylene (20) sorbitan monooleate) or polyoxyethylene-pt-octylphenol (Triton X-100, CAS No. 9002-93-1), under appropriate conditions. An example of a solvent-detergent treatment step is carried out in the presence of 1% (weight / volume) of Polysorbate 80 and 0.3% (v / v) of TnBP for at least 7 hours at 25 ± 1 ° C. The viral inactivation step may also be carried out by treatment with a single detergent, such as Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) or polyoxyethylene-pt-octylphenol (Triton X-100, CAS RN 9002). -93-1). An example of such treatment is an incubation for 30 to 120 minutes (especially for about 1 hour) at a temperature of 20 to 25 ° C (especially at a temperature of about 21 or 22 ° C) in a medium. comprising 0.5 to 2% (v / v) (especially about 1% v / v) of polyoxyethylene-p-octylphenol (Triton X-100, CAS No. 9002-93-1). Step c) Step c) is aimed at improving the purification of the monoclonal antibody or the Fc fusion protein by removing various contaminants, such as residual proteins or nucleic acids from the production host, protein A may have been salted out in step a) or the solvent and / or detergent may have been used in step b). Step c) of the process according to the invention is a cation exchange chromatography step on a resin having a reticulated agarose gel matrix on which sulphonate (-503-) groups are grafted via spacer arms based on dextran. Indeed, the inventors have demonstrated that the use of such a resin makes it possible to increase the load of monoclonal antibody or Fc fusion protein that can be treated at one time, thereby reducing purification costs. (see Example 2). In this step, the monoclonal antibody or Fc fusion protein composition resulting from the viral inactivation step b) is applied to a resin having a reticulated agarose gel matrix on which sulfonate groups are grafted ( -503-) via dextran-based spacer arms.
[0030] The conductivity and / or the pH of the composition resulting from the viral inactivation step b) can advantageously be adjusted before application to the resin. In particular, the conductivity can be adjusted to a value between 3 and 7 mS / cm, in particular between 4 and 6 mS / cm and in particular about 5 mS / cm. The adjustment of the conductivity can in particular be carried out by adding a suitable quantity of purified water, a sodium acetate buffer or, preferably, a formate buffer. The pH can be adjusted to a value between 5 and 7, especially between 5.5 and 6.5, and in particular about 6.0. The adjustment of the pH can in particular be carried out by adding a suitable quantity of 0.5M sodium hydroxide (NaOH). The cross-linked agarose gel, onto which sulfonate groups (-503-) are grafted via dextran-based spacer arms used in step c), can advantageously be in the form of beads having an average diameter. between 10 and 200 [Inn, advantageously between 50 and 150 [Inn, and in particular about 90 [Inn. Examples of crosslinked agarose gel matrices on which sulfonate (-503-) groups are grafted via spacer arms include the following matrices: CaptoTM S (agarose gel matrix) crosslinked, on which sulphonate groups (-503-) are grafted via dextran-based spacer arms in the form of beads with an average diameter of 90 [Inn, marketed by GE Healthcare Life Sciences), Fractogel® EMD 503 "(polymeric matrix of methacrylate, on which are grafted sulfonate groups (-503-) via long chains of linear polymer of acrylamide comprising 15 to 50 units of acrylamide, in the form of beads of a mean diameter of 30 (type S) or 65 (type M) [Inn), and Eshmuno®S (crosslinked polyvinylether hydrophilic matrix, on which are grafted sulfonate groups (-SO3-) via spacer arms, 10 in the form of balls of a d Mean iameter 75-95 [Inn). Advantageously, the resin having a reticulated agarose gel matrix on which are grafted is chosen from a reticulated agarose gel matrix, onto which sulphonate groups (-SO 3 -) are grafted via spacer arms. Dextran base in the form of beads with an average diameter of 90 [Inn (CaptoTM S resin in particular), a methacrylate polymer matrix, on which sulphonate groups (-SO 3 -) are grafted via long chains. of linear acrylamide polymer comprising 15 to 50 units of acrylamide, in the form of beads having an average diameter of 30 (type S) or 65 (type M) [Inn (Fractogel® EMD 503 "resin in particular) and a matrix of crosslinked hydrophilic polyvinyl ether, on which sulphonate groups (-SO 3 -) are grafted by means of spacer arms, in the form of beads with an average diameter of 75-95 [Inn (Eshmuno®S resin in particular), more advantageously the resin is a matrix of crosslinked agarose gel, on which sulphonate groups (SO 3 -) are grafted by means of dextran-based spacer arms in the form of beads with an average diameter of 90 [Inn (CaptoTM S resin in particular).
[0031] The elution can in particular be carried out by increasing the conductivity and / or the pH. In particular, the elution buffer may have a conductivity of between 16 and 20 mS / cm, in particular between 17 and 19 mS / cm and in particular about 18 mS / cm. The elution buffer may have a pH of between 6 and 8, in particular between 6.5 and 7.5, and in particular of around 7.0. It may especially be a 20 mM Tris buffer, NaCl qsp conductivity 18 mS / cm and pH 7.0. The flow rate of the chromatography step is advantageously adjusted to a value corresponding to a residence time of between 1 and 3 minutes, advantageously between 1.5 and 2.5 minutes and in particular of about 2 minutes. Depending on the gel volume, the appropriate flow rate can be calculated based on the following formula: flow rate (mL / min) = gel volume (mL) / residence time (min). Step d) The step d) of the process according to the invention aims to further improve the purification of the monoclonal antibody or of the Fe fusion protein by eliminating various contaminants, such as proteins or residual nucleic acids from the host. of production, the protein A may have been salted out in step a) or the solvent and / or detergent may have been used in step b). It is particularly effective at removing residual nucleic acids. This is an anion exchange chromatography step on a hydrophilic membrane of polyethersulfone coated with a crosslinked polymer on which are grafted quaternary amine groups (Q). The membrane advantageously has an average pore size of between 0.5 and 1 [Inn, advantageously between 0.6 and 0.9 [Inn, between 0.7 and 0.9 [Inn, and in particular about 0.8 [Inn. The membrane advantageously comprises several layers of polyethersulfone 10 coated with a crosslinked polymer on which are grafted quaternary amine groups (Q), advantageously between 10 and 20 layers, especially between 14 and 18 layers, and in particular 16 layers. An example of such a membrane is the Mustang ® Q membrane (16-layer polyethersulfone hydrophilic membrane having an average pore size of 0.8 [Inn, coated with a crosslinked polymer grafted with quaternary amine groups ( Q)) marketed by Pall. In this step d), the monoclonal antibody or Fc fusion protein composition resulting from step c) of cation exchange chromatography is applied to a hydrophilic polyethersulfone membrane coated with a crosslinked polymer on which are grafted quaternary amine groups (Q). The conductivity and / or the pH of the composition resulting from step c) of cation exchange chromatography can advantageously be adjusted before application to the membrane. In particular, the conductivity can be adjusted to a value between 8 and 12 mS / cm, especially between 9 and 11 mS / cm and in particular about 10 mS / cm. The adjustment of the conductivity can in particular be carried out by adding a suitable quantity of a 20 mM phosphate buffer or, advantageously, a 20 mM Tris buffer. The pH can be adjusted to a value between 6 and 10, especially between 7.0 and 9.0, between 7.5 and 8.5, and in particular about 8.0. In particular, pH adjustment can be achieved by adding an appropriate amount of 0.5M sodium hydroxide (NaOH). The membrane is advantageously equilibrated with a Tris buffer, in particular a Tris buffer having the following characteristics: a concentration of between 15 and 25 mM, between 16 and 24 mM, between 17 and 23 mM, between 18 and 22 mM, between 19 and 25 mM; and 21 mM, in particular about 20 mM, pH between 6 and 10, between 7.0 and 9.0, 7.5 to 8.5, in particular about 8.0, a conductivity of between 8 and 12 mS / cm, especially between 9 and 11 mS / cm and in particular about 10 mS / cm.
[0032] Step e) Step d) of the method according to the invention aims to eliminate viruses, and in particular small non-enveloped viruses more resistant to viral inactivation treatments, likely to be in the purified antibody composition or Fe fusion protein to ensure viral safety of the final drug product. Indeed, conventional viral inactivation treatments, and in particular the solvent-detergent or detergent-only treatment, have limited effectiveness with respect to non-enveloped viruses, such as parvoviruses or hepatitis A virus. However, nanofiltration, which relies on an exclusion mechanism depending on the size of the particles, is known to be effective on non-enveloped viruses. The most common filters used to exclude small non-enveloped viruses are Planova® filters marketed by Asahi Kasei, in particular Planova® 15N and Planova® 20N filters, with average pore sizes of 15 and 19 nm, respectively. These filters, consisting of a cuprammonium-regenerated cellulose hollow fiber membrane, are characterized by low pore size dispersibility (± 2 nm around the average size). However, these filters are very expensive and do not allow the treatment of a high protein load in a limited period of time (eg acceptable treatment time of 4 hours), unless significantly increase the filtration area (and therefore in fine the cost of this step).
[0033] In the context of the present invention, the inventors have demonstrated that it is very advantageous to use a Viresolve® Pro filter (filter having an asymmetric double polyethersulfone membrane retaining at least 4 decimal logs of virus having a size of at least 20 nm) instead of a Planova® 15N or Planova® 20N filter, the Viresolve® Pro filter allows nanofiltration of a much larger antibody load than the Planova® 15N and Planova® 20N filters (see Example 4). Step e) therefore consists of a nanofiltration step with a filter having a double polyethersulfone membrane with a porosity of about 20 nm. Such filters include, in particular, the Viresolve® Pro filter (a filter having an asymmetric polyethersulfone double membrane with a porosity of about 20 nm, marketed by Merck-Millipore) and the Virosart® CPV filter (a filter having a symmetrical double-walled membrane). polyethersulfone with a porosity of about 20 nm, marketed by Sartorius). The nanofiltration of step e) is advantageously carried out using a filter having an asymmetric polyethersulfone double membrane with a porosity of about 20 nm, such as the Viresolve® Pro 35 filter marketed by Merck-Millipore. By a porosity of about 20 nm, it is meant that the average pore size of the filter is between 17 and 25 nm, advantageously between 17 and 24 nm, between 17 and 23 nm, between 17 and 22 nm, between 17 and 25 nm, and 21 nm, 17 to 20 nm, 18 to 25 nm, 18 to 24 nm, 18 to 23 nm, 18 to 22 nm, 18 to 21 nm, 18 to 20 nm, 19 to 25 nm, between 19 and 24 nm, between 19 and 22 nm, between 19 and 22 nm, between 19 and 21 nm, between 19 and 20 nm, between 20 and 25 nm, between 20 and 24 nm, between 20 and 23 nm , between 20 and 22 nm, or between 20 and 21 nm.
[0034] In an advantageous embodiment, step e) further comprises a preliminary filtration step through a depth filter comprising cellulose fibers, diatomaceous earth and a negatively charged resin (pre-filter Viresolve PreFilter). or VPF) or a polyethersulfone membrane having a porosity of 0.22 μm functionalized with SO 3 - groups (pre-filter Viresolve pro Shield in particular) Optional steps The method according to the invention may further comprise a step of ultrafiltration and / or diafiltration, which can be between the step d) of anion exchange chromatography and the nanofiltration step e), or after the step e) of nanofiltration. carried out using centramate type cassettes 50 kDa (marketed by Pall) or Pellicon 2 (sold by Merck Millipore) with a dialysis buffer comprising polysorbate 80 in the case where the Rinse is after step e) nanofiltration.
[0035] In addition, one or more sterilizing filtration steps through filters having a porosity of about 0.1 to 0.5 μm (especially about 0.2 μm) may be present at different stages of the process according to the invention. the invention. These steps can in particular be carried out using a Millipak filter of 0.22 μm.
[0036] The following examples are intended to illustrate the present invention. EXAMPLES Example 1: Optimization of Protein A Affinity Chromatography Step a) The protein A affinity chromatography step is an indispensable step in the purification of antibodies, but is also the step the most expensive of antibody purification methods. In order to significantly reduce the cost of this step while maintaining the purity and quality of the product, the inventors tested different protein A affinity chromatography resins and different elution buffers, and measured the influence of the protein. resin and elution buffer on a number of parameters. Materials and Methods Comparison of Four Protein A Affinity Chromatography Resins Columns Tested and Prepared The characteristics of the columns tested were: Column Lot Volume Size Resin Size (mL) (cm) Mean Agarose Beads MabSelect SuReTM 10111269 4 , 7 4.7 x 0.77 strongly 85 [Crosslinked poly (styrene-divi-n-ylbenzene) Innate coated Poros GoPureTM 121004 5.655 1.2 x 5 Polymer 45 [Cross-linked polyhydroxy Inn Toyopearl AF-Polymer rProtein A-650F 0022810 5.02 14.6 x 3 methacrylic 45 [Inn Amsphere Protein 10000006- polymer 5 11.3x 5 49 [Inn A JWT203 methacrylic CO3 Table 1. Characteristics of columns tested The columns are sanitized according to the following sequence: Solution Volume column Flow (mL / min) ) (VC) Purified water 2 3 NaOH 0.5 M 5 3 then 30 min contact time Purified water Qsp pH <8.0 3 NaCl 2M 5 3 Table 2. Sanitization sequence of the columns Determination of the "Breakt point" hrough "A filtered thawed antibody solution 0.2 [Inn at 2.3 g / L is injected into the chromatography apparatus (Akta Basic) without passing through the column. The OD 280 nm thus read corresponds to the maximum 280 nm OD. The latter is 664 mAU. The point corresponding to 10% of a pressure loss of the column, called - Breakthrough "(10% 8T), is thus determined at 66.4 mAU. The UV cell and the device circuit are then rinsed with water and then with equilibration buffer. Determination of the dynamic binding capacity at 10% passage (DBCiomer) Once connected, the column is equilibrated with Buffer A (25 nM Tris, 25 nM NaCl, 5 nM M EDTA, pH 7.1). When the column is equilibrated, a wash step is performed with the antibody solution to fill the pipes upstream of the column. Regardless of the column, the 0.2 micron filtered defrosted antibody solution is injected at a flow rate of 3 mL / min (ie a residence time of about 1.6 min) and a follow-up of the 280 nm OD is performed. .
[0037] Comparison of Four Elution Buffers Four elution buffers of Protein A affinity chromatography were tested at different concentrations and pH, and their impact on the eluate appearance and percentage of eluate. Monomeric IgG (and thus on the presence of aggregates) was analyzed. Composition of Buffers Tested The four buffers tested were as follows: Buffer Composition Test tested pH concentrations Citrate Trisodium citrate 5 to 25 mM 2.6 to 3.6 dihydrate Maleate Maleic acid, 5 to 25 mM 2.6 to 3.6 NaOH 0 5 M Acetate Sodium acetate 5 to 25 mM 2.6 to 3.6 dihydrate Sodium formate 5 to 25 mM 2.6 to 3.6 Table 3. Buffers tested Eluate appearance analysis 15 Once the eluate has been neutralized, a visual analysis is performed by the operator with the following scale of assessment: 0- Clear appearance; 1- Light apparent disorder; 2-Medium disorder; 3- Strong disorder (Opalescence). The neutralized eluate is then stored at room temperature for 1 hour. A second observation is conducted by the same operator on the same scale of appreciation.
[0038] Analysis of the percentage of monomeric IgG A volume of 500 μL from the neutralized eluate is analyzed by HPLC-SEC (High Performance Liquid Chromatography - Size Exclusion Chromatography) on a column of Superose 12. The peaks generated by reading the density optical at 280 nm are integrated by Breeze software and areas converted into percentages.
[0039] Results Comparison of Four Protein A Affinity Chromatography Resins The results obtained for the four columns tested are shown in Figure 1.
[0040] Figure 1A shows that for the MabSelect SuReTM column, a volume of 47 mL was injected onto the column until a 280 nm OD of 66.4 mAU was obtained. The column fixation capacity at 10% of BT (DBCiomyr) is therefore 23 mg / mL of MabSelect SuReTM gel. FIG. 1B shows that for the Poros GoPureTM column, a volume of 95 mL was injected onto the column until obtaining a 280 nm OD of 66.4 mAU. The column fixation capacity at 10% of BT (DBCio% Br) is therefore 38.64 mg / mL of Poros GoPureTM gel. FIG. 1C shows that for the Toyopearl AF-rProtein A-650F column, a volume of 80 mL was injected onto the column until an OD 280nnn of 66.4 mAU was obtained. The column fixation capacity at 10% BT (DBC10% 13-r) is therefore 36.65 mg / mL of Toyopearl AF-rProtein A-650F gel. Figure 1D shows that for the AmsphereTM Protein A JWT203 column, a volume of 93.4 mL was injected onto the column until a 280 nm OD of 66.4 mAU was obtained. The column fixation capacity at 10% of BT (DBC10, BT) is therefore 42.95 mg / mL of AmsphereTM Protein A gel JWT203. Table 4 below summarizes the data obtained with the four columns and shows that the Poros GoPureTM columns (crosslinked poly (styrene-divinylbenzene) matrix coated crosslinked polyhydroxylated polymer), Toyopearl AF-rProtein A-650F 25 (methacrylic polymer matrix). ), and AmsphereTM Protein A JWT203 (methacrylic polymer matrix) accept a significantly higher antibody load than the MabSelect SuReTM column (highly crosslinked agarose matrix). Column Volume injected at DBC10% Br 90% of DBCio% Fr 10% of OD max (in mg / ml of (in mg / ml of (10% 8T) gel) gel) MabSelect SuReTM 47 23 21 Poros GoPureTM 95 38.64 35 Toyopearl AF-rProtein A- 80 36.65 33 650F AmsphereTM Protein A 93.37 42.95 39 JWT203 Table 4. Antibody load accepted by different columns tested.
[0041] In order to confirm the maximum load of the three columns allowing the highest antibody load, filtered clarified supernatant was injected onto each column with an antibody charge equal to 90% of the value of DBCiomyr. The charges thus applied are as follows: Column 90% DBCio% Fr Poros GoPureTM 35 mg / mL AF-rProtein A-650F 33 mg / mL AmsphereTM Protein A JWT203 39 mg / mL 5 Table 5. Charges applied to the different columns Two tests were performed for each of the three columns tested. The results obtained are summarized in Table 6 below and show that the AmsphereTM Protein A JWT203 column gives the best results, both in terms of antibody load and purity, appearance of the eluate, and even pH of the eluate (which is then adjusted to a pH of about 6.0 for the rest of the process). The performance of this column is otherwise similar to that of the other columns. Although the antibody load accepted by the Toyopearl AF-rProtein A-650F column is slightly lower than that of the AmsphereTM Protein A JWT203 column, it nevertheless allows an antibody load greater than 30 mg / mL of gel and gives satisfactory results in terms of purity, appearance of the eluate, and pH of the eluate. Although allowing a high antibody load (at least 35 mg / ml of gel), the Poros GoPure ™ column gives rise to a poor behavior of the eluates 20 which have all appeared turbid. In addition, the results are poorer in terms of yield and purity.
[0042] 3025515 27 Column Injection * Test Volume, Appearance of Eluate pH Efficiency Purity (mg / ml eluate gel eluate) Poros GoPureTM 35 mg / mL 1 3.3 Disorder 3.52 95.23 83.95% 2 2.7 Very 3.75 89 , 92.52% disorder Toyopearl AF-33 mg / mL 1 3.2 Clear 3.82 94.48 94.00% rProtein A-650F 2 2.2 Opalescent 4.88 100 93.13% AmsphereTM 39 mg / mL 1 3.2 Clear 4.36 91.96 95, 48% Protein A JWT203 2 3 limpid 4.78 97.5 95.88% * Charge corresponding to 90% of Dynamic Binding Capacity (DCB) at 10% BT (Breakthrough) Table 6. Comparison of three columns of affinity chromatography at the Protein A Compared with the MabSelect SuReTM gel commonly used in the first step of antibody purification by protein affinity chromatography, Annsphere Protein A gel JWT203 is presented as a product having a price per liter twice cheaper and a load capacity about twice as much. This column thus makes it possible to reduce by four the cost of the first purification step by protein affinity chromatography.
[0043] Comparison of four elution buffers The results concerning the appearance of the eluate are shown in FIG. 2 and show that the maleate and formate buffers make it possible to obtain a clear or slightly opalescent stabilized neutralized eluate at concentrations of between 5 and 5. and 10 mM (particularly at 5 mM) and a pH between 2.6 and 3.6.
[0044] The acetate buffer does not make it possible to obtain clear eluates and can even lead, at low pH and medium molarity, to highly disturbed eluates 1 hour after neutralization. As for the citrate buffer, the results are very bad in terms of appearance for the stabilized neutralized eluate.
[0045] The results concerning the percentage of monomeric forms of the antibody in the neutralized eluate are shown in FIG. 3, and show that more than 98% of the antibodies are in nonnormeric form in the neutralized eluate after elution with a citrate buffer. formate at a molarity of 5 mM and at a pH between 2.6 and 3.6.
[0046] Although allowing the eluates to be clear or only slightly opalescent, the maleate buffer leads to a significant formation of antibody aggregates (always more than 5%), which is undesirable. As for the acetate buffer, the results are less good than with a citrate or formate buffer.
[0047] In total, the buffer giving the best results, both in terms of appearance of the neutralized eluate and percentage of monomeric antibody forms in the neutralized eluate, is the formate buffer, preferably used at a molarity of 5. at 10 mM (preferably 5 mM) and at a pH between 2.6 and 3.6 (especially at a pH of 3.1).
[0048] Conclusions In comparison with the MabSelect SuReTM gel commonly used in the first antibody purification step by protein A affinity chromatography, the inventors have been able to select a column allowing to reduce by four the cost of the first purification step. by affinity chromatography to protein A, while maintaining the purity and quality of the purified product. In addition, the inventors have also selected an elution buffer of particular interest to guarantee a neutralized eluate which is clear or slightly opalescent and comprises a very large majority of nonnonneric forms of the antibody. The specific combination of column and buffer selected by the inventors 20 thus makes it possible to greatly reduce the cost of the purification while guaranteeing a product of high purity and quality. Example 2 Optimization of cation exchange chromatography step c) A virally inactivated eluate from step b) of the method was purified by cation exchange chromatography on two distinct column types: an SP Sepharose column and a CaptoTM S. column Materials and methods SP Sepharose® cation exchange chromatography A virally inactivated eluate from step b) of the method was adjusted to 50 mosm / kg and pH 7.2, and was injected onto an SP column. Sepharose® at a loading of approximately 30.5 g / L of gel according to the sequence: 3025515 29 Denomination Solution / Buffer Flow Volume Parameters controlled used buffer minimum Elimination of purified water s 200 cm / h 2VC NA storage solution Balancing Phosphate Buffer of 200 cm / h 5VC PH and 20 mM sodium; pH 7.2; 50 mosm / kg osmolality Injection Inactivated product adjusted to 50 mosm / kg and pH 7.2 s 200 cm / h About 5 liters NA Return to baseline Buffer Phosphate of 200 cm / h 5VC sodium DO 20 mM; pH 7.2; 50 mosm / kg Reversal of the flow on the column Elimination of the phosphate buffer of 200 cm / h 14VC NA solvent - sodium 20 mM; pH 7.2; 50 mosm / kg detergent Elution Buffer Phosphate of 200 cm / h 4VC Harvest at OD 0.2AU sodium 20 mM; 150 mM NaCl; pH 7.2; 50 mg / kg Table 7. Sequence of purification of a virally inactivated eluate from step b) of the process according to the invention by cation exchange chromatography on SP Sepharose® cation exchange chromatography on CaptoTM S 5 the test, in parallel with SP Sepharose® A virally inactivated eluate from step b) of the process was adjusted to 5.09 mS / cm by adding 5mM sodium acetate buffer pH6.0, and pH 5.98 , and was injected onto a CaptoTM S column at a load of approximately 67.3 g / L of gel according to the sequence: 3025515 Denomination Solution / Buffer Flow Minimum buffer volume Parameters controlled Elimination of purified water 8 mL / min That is 2VC NA solution of 240 cm / h storage Residence time of 3 min Equilibration Sodium acetate 20mM; Up to pH equilibration and conductivity NaCI qsp conductivity 5mS / cm; pH 6.0 Injection Eluate Inactivated Protein A 365 mL OD 280 nm (67.3 g / L) Back to the line Sodium Acetate 20mM; 2VC OD 280 nm base NaCI qsp conductivity 5mS / cm; pH 6.0 Injection direction Flow reversal Wash 20 mM Sodium acetate; 8 mL / min That is 14VC OD 280nm NaCl qsp conductivity 240 cm / h 5mS / cm; pH 6.0 Elution Tris 20mM; NaCI 10VC Harvest at qsp conductivity 18mS / cm; 250 mAu DO 280 nm pH 7.0 Table 8. Sequence of purification of a virally inactivated eluate from step b) of the process according to the invention by cation exchange chromatography on CaptoTM S 2nd test A virally inactivated eluate from step b) of the process was adjusted to 5.04 mS / cm by adding 103.8 mL of purified water and pH 6.02 by addition of 0.5 M NaOH. For this step , Capto S gel is compressed in a column of 1 cm in diameter thus obtaining a column volume of 4.8 mL for a height of 6.1 cm. The injection on the Capto S cation exchange column proceeded as follows: 3025515 31 Name Solution / Buffer Flow Volume Minimum parameters of buffer / solution controls Elimination of purified water s 600 2VC NA solution of cm / h Storage Balancing Phosphate 20mM sodium; s 600 Up to pH and NaCl qsp conductivity 5mS / cm; pH 6.0 cm / h equilibrium conducti. Injection Eluat Protein A s 600 Based on [] in OD 280nm cm / h IgG Back to Baseline Sodium Phosphate 20mM; s 600 2VC OD 280 nm NaCl qsp conductivity 5mS / cm; pH 6.0 cm / h Direction of injection Flow reversal Wash Sodium phosphate 20mM; s 600 14VC OD 280nm NaCl qsp conductivity 5mS / cm; pH 6.0 cm / h reverse flow, downflow Elution Sodium phosphate 20mM; s 400 10VC NaCl harvest qsp conductivity cm / h 250mAu OD 18mS / cm; pH 6.9 280 nm Table 9. Sequence of purification of a virally inactivated eluate from step b) of the process according to the invention by cation exchange chromatography on CaptoTM S in a second test.
[0049] The column is loaded with 120 grams of IgG / L gel to determine the maximum capture capacity of the Capto S gel. Determination of the Breakthrough Point and Dynamic Attachment Capacity at 10% Passage (DBC10%, 137-) A virally inactivated eluate from step b) of the method was adjusted to 5.05 mS / cm and pH 6.04 by addition of 6N HCl and EPA ( Pyrogen Purified Water). For this step, Capto S gel is compressed in a column 0.5 cm in diameter thus obtaining a column volume of 3.8 mL for a height of 19.5 cm. The antibody solution is then injected into the chromatography apparatus (Akta Basic) without passing through the column. The OD 280 nm thus read corresponds to the maximum OD 280 nm. The UV cell and the device circuit are then rinsed with water and then with equilibration buffer. The dynamic binding capacity at 10% passage (DBC10% 13-r) was then determined for three residence times (1, 2 and 3 minutes) by injecting on the column the antibody solution at different flow rates ( 3.8 mL / min, 1.9 mL / min, and 1.3 mL / min, respectively) and following OD 280. Results Comparison of SP Sepharose® cation exchange chromatography and 5 cation chromatography on CaptoTM S SP Sepharose®: the collected eluate volume is 150 ml, with an estimated protein concentration of 25.96 g / L and an osmolality of 273mosm / kG and a pH of 7.09. The amount of antibody present in the eluate was 3894 mg. The yield of step 10 is 88.6%. The product is clear with some particles. CaptoTM S: at the end of elution, a volume of 174 mL is obtained, ie 7.3 VC. The final concentration of the eluate is 9.0 g / L and limpid appearance at the column outlet. The stage yield is then 94.3% and the pH and the conductivity of the eluate are respectively 6.66 and 17.11 mS / cm.
[0050] The eluate is then filtered through a 0.22 micron Millipak capsule previously conditioned with 20 nM Tris buffer; NaCI qsp conductivity 18 mS / cm; pH 7.0. The filtration is carried out with the L02 pump pump at a speed of 30 rpm via a pipe 184. The filter is then rinsed with 20 nM Tris buffer; NaCI qsp conductivity 18nnS / cnn; pH 7.0. At the end of this filtration, a volume of 208.8 ml is obtained with a concentration of 6.8 g / l and a filtration stage yield of 90.7%. The purity of the filtered eluate is 99.6% (in proteins). Parameter analyzed SP Sepharose® CaptoTM S Antibody load 30.5 g / L gel 67.3 g / L gel Eluted volume 150 mL 174 mL Yield 88.6% 94.3% Aspect of the clear eluate with limpid few particles Purity ND 99.6% Table 10. Comparative data for purification by cation exchange chromatography on SP Sepharose® and CaptoTM S.
[0051] Table 10 above shows that the CaptoTM S column allows twice the antibody loading of the SP Sepharose® column (resulting in lower costs), better yield (hence cost reduction). , while guaranteeing a limpid appearance and a very good purity. The second point of the curve of OD is 110 nm, the OD 280 nm which up to that point was 1650 mAU rises progressively, expressing an IgG leak through This gives a maximum fixing capacity of about 84 g / L.
[0052] At the end of elution, a volume of 57.2 mL is obtained, ie 11.9 VC. The elution is at first very fast but then drags in time (very slow down of the D0280nm). The final concentration of the eluate is 7.30 g / L and limpid appearance at the column outlet. On the other hand, particles appear after a few minutes. The stage yield, based on the 110 mL of product injected, is 100%.
[0053] In addition, after Capto S gel cation exchange chromatography, the IgG solution appears 100% pure (in proteins). Thus, this second test confirms that the maximum antibody load of the CaptoTM S column is much greater than that of the SP Sepharose® column. Breakthrough Point (BT) and Dynamic Capillary 10% Passage Capability (DBCiomyr) of the CaptoTM S Column The maximum OD 280nnn - read by injecting the antibody solution into the chromatography apparatus (Akta Basic) without going through the column - is 535 mAU. The point corresponding to 10% of a pressure loss of the column, called - Breakthrough "(10% 8T), is thus determined at 54 mAU.
[0054] Measurement of the dynamic attachment capacity at 10% passage (DBC10% 13-r) for residence times of 1, 2 and 3 minutes gave the results shown in Table 11 below: Residence time Volume injected at DBC10% 13-r 90% of DBCimBT (flow rate) 10% of OD max (in g / L of gel) (in g / L of gel) (10% 8T) 1 minute 207.8 87 78 (3.8 mL) / min) 2 minutes 250.5 104 94 (1.9 mL / min) 3 minutes 265.6 111 100 (1.3 mL / min) Table 11. Measurement results of dynamic attachment capacity at 10% passage (DBCiomyr) for residence times of 1, 2 and 3 minutes. These results confirm the much higher binding capacity of the CaptoTM S column compared to the SP Sepharose® column.
[0055] 3025515 34 Conclusions The selection by the inventors of the CaptoTM S column for step c) of cation exchange chromatography, in place of the SP Sepharose® column commonly used in this antibody purification step, again allows 5 to reduce the costs of purification. Example 3: Optimization of the e) Nanofiltration Step The e) nanofiltration step is essential to ensure the viral safety of the final antibody composition, particularly vis-à-vis small non-enveloped viruses. However, this step is also a very expensive step, nanofilters being very expensive products. Since each nanofilter is used only once, the inventors have tested several distinct nanofiltrées in order to optimize the load of antibodies that can be treated at one time, so as to reduce the cost of this step. In addition, the interest of using a prefilter with greater porosity to increase the antibody load has also been studied. Materials and Methods Comparison of Three Separate Filters Starting Material The mustang filtrate Q (step d) is diluted 2/3 with trisodium citrate buffer dihydrate 22.05 g / L; NaCl 18.23 g / L; pH 6.5; 800 mosm / kg is a final volume of 555mL. A 0.2 μm filtration is then carried out on a Millipak 40 filter of 0.02 m 2 followed by rinsing with buffer K, ie a final volume of 672 ml, a concentration of 5.28 g / l, a pH of 7.14 and an osmolality. of 367 mosm / kg. The product is then filtered through a 0.1 μm hydrophilic PVDF pall filter, followed by a K buffer rinse, a final volume of 643 ml, a concentration of 5.16 g / l, a pH of 7, 12 and an osmolality of 366 mosm / kg. Filters used The characteristics of the nanofilters tested are the following: Filter Manufacturer Membrane Porosity Planova® 15N Asahi Kasei hollow fiber 15 ± 2 nm cellulose regenerated with cuprammonium Planova® 20N 19 ± 2 nm Viresolve pro 20N Millipore double membrane About 20 nm asymmetric polyethersulfone Table 12. Characteristics of the filters tested 3025515 Nanofiltration on Planova® 15N The nanofiltration step is carried out on Planova® 15N filter of 0.001 m 2 balanced in trisodium citrate buffer 7.35 g / L; NaCl 9 g / L; pH 6.5; 360 mosm / kg at a pressure of 300 ± 50 mbar. The average flow rate was 0.15nnUnnin. The filtrate was clear at the end of nanofiltration. Nanofiltration on Planova® 20N The nanofiltration step is carried out on Planova® 20N filter of 0,001m2 balanced in trisodium citrate 7.35g / L buffer; NaCl 9 g / L; pH 6.5; 360 mosm / kg at a pressure of 800 ± 50 mbar.
[0056] Nanofiltration on Viresolve pro 20N The nanofiltration step is carried out on a Viresolve pro 20N filter of 3.1 cnn 2 equilibrated in trisodium citrate buffer 7.35 g / L; NaCl 9 g / L; pH 6.5; 360 mosm / kg at a pressure of 2 bar. Validation of the use of the Viresolve pro 20N filter The Viresolve pro 20N filter was tested on several purified antibody compositions in order to validate the antibody load capable of being filtered at one time. Test 1 The entire cation exchange chromatography eluate (step c) of the process according to the invention) was filtered on a 0.22 μm Mini Kleenpak capsule with a filter rinse using phosphate buffer. 20 nnM; pH 6.9; conductivity 5 mS / cm. At the end of this filtration, a volume of 64.3 ml is obtained with a concentration of 5.9 g / l. The filtration yield is 90.7%, this is explained by the fact that the filter was rinsed with little buffer to avoid going down too low in IgG concentration for the next nanofiltration step. The product is stable after 0.22 filtration [Inn. It is then stored 24h at + 4 ° C. The 64.3 ml of starting material were injected at 2 bars onto the Viresolve Pro + nanofilter of 3.1 cnn 2 equilibrated with 20 nnM sodium phosphate buffer; conductivity of 5 mS / cm; pH 6.9. The appearance of the product stored 24h + 4 ° C before nanofiltration was clear. The nanofiltration step was therefore carried out directly on the unfiltered product over 0.1 μm beforehand. Test 2 The appearance of the product resulting from the anion exchange chromatography on Mustang Q (step d) of the process according to the invention), subjected to a dialysis step, and stored 24h at + 4 ° C. was clear. Prior to nanofiltration, the latter was filtered through a 0.1 μm Mini Kleenpak capsule with a filter rinse using trisodium citrate buffer 30.5% dihydrate 8.82 g / L, 3.25 g / L NaCl; Mannitol 17 g / L; pH 6.5; 300 mosm / kg. At the end of this filtration, a volume of 264 ml is obtained with a concentration of 5.1 g / l. The filtration yield is 100%. The filtered dialyzed solution is injected at 2 bar onto the Viresolve® Pro nanofilter of 3.1 cnn 2 previously equilibrated in trisodium citrate dihydrate buffer 8.82 g / l, NaCl 3.25 g / l; Mannitol 17 g / L; pH 6.5; 300 mosm / kg. Test 3 The Mustang Q® eluate (step d) of the process according to the invention) is injected at 2 bars onto the Viresolve® Pro nanofilter of 3.1 cnn 2 equilibrated beforehand in 20 nM Tris buffer; PH 6.5; 10 mS / cm. Use of a Pre-Filter The nanofiltration of the same Mustang® Q eluate (step d) of the process according to the invention) directly on the Viresolve® Pro filter, or after passing through a Sartorius® or Millipore® pre-filter was tested.
[0057] Direct Nanofiltration The Mustang® Q eluate (step d) of the process according to the invention) is injected at 2 bars onto the Viresolve® Pro nanofilter of 3.1 cnn 2 equilibrated beforehand in 20 nM Tris buffer; pH 6.5; 10 mS / cm. This experiment corresponds to the above test 3 of the Viresolve® Pro filter.
[0058] 20 Nanofiltration after passing through a Sartorius® pre-filter A series assembly of a Sartorius® pre-filter on the Viresolve® Pro nanofilter is performed. The Mustang® Q eluate (step d) of the process according to the invention) is injected at 2 bar the Viresolve® Pro nanofilter of 3.1 cnn2 previously equilibrated in Tris 20 nnM buffer; pH 6.5; 10 mS / cm.
[0059] Nanofiltration after passing through a Millipore® prefilter A series assembly of a Millipore® prefilter (optiscale®-40 Viresolve prefilter ref SSPVA4ONB9) on the Viresolve® Pro nanofilter of 3.1 cnn2 is carried out. The Mustang® Q eluate is injected at 2 bars onto the Viresolve® Pro nanofilter of 3.1 cnn 2 previously equilibrated in 20 nM Tris buffer; pH 6.5; 10 mS / cm.
[0060] Nanofiltration after passing through a Millipore® pre-filter (validation on a second product) A series assembly of a Millipore prefilter (Viresolve Pro ref C2NA74678) on the Viresolve Pro nanofilter of 3.1 cnn2 is carried out. The product resulting from the anion exchange chromatography on Mustang Q (step d) of the process according to the invention) and from the dialysis step is injected at 2 bars on the Viresolve Pro + assembly of 3.1 cnn2 equilibrated 3025515 37 previously with trisodium citrate dihydrate buffer 8.82 g / L, NaCl 3.25 g / L; Mannitol 17 g / L; pH 6.5. Nanofiltration after passing through a Millipore® prefilter (validation on a second product) A parallel assembly of two Millipore prefilters (Viresolve Pro Shield ref. 5 C2NA74678) is upstream of the 3.1 cm2 Viresolve Pro nanofilter. The assembly is pre-equilibrated in its entirety under a pressure of 2 bars with EPA then in buffer Trisodium citrate dihydrate 8.82 g / L, NaCl 3.25 g / L; Mannitol 17 g / L; pH 6.5. The second prefilter remains clamped at the beginning of the injection of the product, it must be declamped in case of clogging of the first prefilter. This arrangement makes it possible to determine the Vmax of the nanofilter without the prefilter possibly being limiting. Average flow over 10 min at the EPA = 2.18 g / min Average flow over 10 min in Citrate buffer = 2.52 g / min Results Comparison of three distinct filters Nanofiltration on Planova® 15N Figure 4A represents the flow rate of filtration according to the antibody load. The average flow rate was 0.15 mL / min for an antibody load of up to almost 200 g / m2. The filtrate was clear at the end of nanofiltration.
[0061] 20 Nanofiltration on Planova® 20N Figure 4B shows the filtration rate as a function of the antibody load. The average flow rate was 0.8 mL / min for an antibody load of up to almost 1100 g / m2. The filtrate was clear at the end of nanofiltration. Nanofiltration on Viresolve® pro 20N Figure 4C shows the filtration rate as a function of the antibody load. The average flow rate was 2.4 mL / min for an antibody load of up to about 5500 g / m2. The filtrate was clear at the end of nanofiltration. Comparison of nanofiltered antibody load versus filtration time Figure 5 shows the nanofiltered antibody load as a function of filtration time, and very clearly illustrates the strong superiority of the Viresolve® pro 20N filter over Planova filters. ® 15N and Planova® 20N.
[0062] By extrapolation, after 4 hours Planova® 15N -> 200 g of Planova® 20N IgG / m2 -> 1000 g of IgG / m2 Viresolve® pro + -> 5000 g of IgG / m2 should be obtained. Validation of the use of the Viresolve pro 20N filter Test 1 A clogging of the Viresolve® Pro + filter is noted after 30 min. A charge of 141 mg of product could thus be nanofiltered over 3.1 cm 2 of filtering surface, ie an amount of antibody of 455 g / m 2 of nanofilter. This load is significantly lower than previously obtained when comparing the three Planova® 15N, Planova® 20N and Viresolve® Pro + filters (4865 g / m2). Nevertheless, the decrease may be related to the characteristics of the filtered product (different from that filtered when comparing the three filters) and the maximum load remains well above that possible with the Planova® 15N filter.
[0063] Test 2 A clogging of the Viresolve® Pro filter is noted after 26.41 ml, ie a quantity of 135.48 mg of antibody and a loading of 437 g / m2 of nanofilter. The charge is about 300 g / m2 at V75 and 425 g / m2 at V90. The maximum load is similar to that obtained in test 1 and confirms the interest of the Viresolve® Pro filter compared to Planova® filters. The purity of the nanofiltered product is determined by size exclusion chromatography (HPSEC). It is estimated at 98.83% at this stage of the process. Run 3 Filtration was stopped following clogging of the nanofilter after 7.21 mL of product, 25.96 mg of antibody and 84 g / m2 load. This maximum load is much lower than those previously observed and illustrates a possible variability of the maximum load depending on the state of the nanofilter product. This variability justifies testing the use of a prefilter. Use of a prefilter Direct nanofiltration Filtration is stopped following the clogging of the nanofilter after 7.21 ml of product, that is to say 25.96 mg of antibody and a charge of 84 g / m 2. Nanofiltration after passing through a Sartorius® pre-filter A clogging of the prefilter is noted at the end of 23.82 ml, ie a quantity of 85.75 mg of filtered antibody and a load of 277 g / m2. In this case, the load limit 3025515 39 is imposed by the prefilter. The Sartorius® prefilter therefore improves the maximum antibody load, but does not restore a very high antibody load. Nanofiltration after passing through a Millipore® prefilter A clogging of the prefilter is noted after 97 ml, ie an amount of 349.2 mg of antibody and a load of 1126 g / m2. In this case, the load limit is imposed by the prefilter. The Millipore®-optiscale®-40 Viresolve® prefilter prefilter therefore improves the maximum antibody load, resulting in a higher than already high load of Validation Tests 1 and 2 described above with the Viresolve® Pro nanofilter alone. .
[0064] Nanofiltration after passing through a Millipore® pre-filter (validation on a second product) A clogging of the assembly is noted after 2 h 25 min after passing 193.2 ml (at 3.23 g / l after filtration 0.22). [Inn) is an amount of about 624 mg of antibody and a load of 2013 g / m2. The assembly is then clamped and put on hold until the next day.
[0065] After replacing the prefilter and resuming the filtration, a clogging of the assembly is noted after 24 min with 14.0 ml, ie a quantity of 45.2 mg of antibody (charge of 146 g / m2). The total load of the Viresolve® Pro + solution is therefore 2159 g / m2. Note that the destabilization of the product during the night does not know which of the prefilter or the filter is at the origin of this clogging.
[0066] In all cases, this experiment validates the advantage of using a prefilter before nanofiltration on the Viresolve® Pro + filter, to guarantee a very high antibody load. Nanofiltration after passing through a Millipore® prefilter (validation on a product Seme) The 418.6 ml of product resulting from dialysis, ie 938 mg (3 kg / m 2), were completely nanofiltered in 217 minutes with a final flow equivalent to 71. 5% of the initial flow. The volume of nanofiltrate obtained was 407.5 ml at a concentration of 2.25 g / l, ie 917 mg of proteins, which gives a no-rinse step yield of the nanofilter of 97.8%. The assembly used made it possible to obtain a load of 2958 g / m2, ie a load 2.6 times greater than that of the first test and 1.4 times greater than that obtained with the 2nd product.
[0067] SUMMARY TABLE OF THE RESULTS OBTAINED The various results obtained are summarized in Table 13 below: Pre-filter Nanofilter Antibody load Comments No Planova® 15N 200 g / m2 Very low No Planova® 20N 1100 g / m2 Medium No Viresolve® pro 20N Comparison 3 Potentially high but very high: 5500 g / m2 Validation: variable, with Test 1: 455 g / m2 Test 2: 437 g / m2 Test: 3: 84 g / m2 sealing problems Sartorius® Viresolve® pro 20N 277 g / m2 Millipore® pre-filter clogging - optiscale0-40 Viresolve® prefilter »Viresolve® pro 20N Test 1 (1 pre-filter): Medium at 1126 g / m2 high, with Test 2 (1 pre-filter): limited variability 2159 g / m2 Test 3 (2 prefilters in parallel): 2958 g / m2 Table 13. Synthesis of the results obtained for step e) nanofiltration.
[0068] Conclusions The experiments carried out by the inventors clearly show the advantage of using a Viresolve® Pro filter for the nanofiltration of a purified antibody composition, in order to greatly increase the load of antibodies treated at one go and thus reduce significantly the costs associated with this particularly expensive step.
[0069] In addition, the addition of a prefilter makes it possible to further improve the treated antibody load at one time. In total, compared to a nanofiltration step using a Planova® 15N filter and an antibody load of 50 g / m2 (Applicant's prior process), the modifications related to the selection of the Viresolve® Pro filter and the addition pre-filter allow to obtain a load gain of a factor greater than 40.
[0070] 3025515 41 BIBLIOGRAPHIC REFERENCES Fahrner RL, Knudsen HL, Basey CD, Galan W, Feuerhelm D, Vanderlaan M, Blank GS. Industrial purification of pharmaceutical antibodies: development, operation, and validation of chromatography processes. Biotechnol Genet Eng Rev. 2001; 18: 301-27.
[0071] Liu HF, Ma J, Winter C., Bayer R. Recovery and purification process development for monoclonal antibody production. MAbs.
[0072] 2010 Sep-Oct; 2 (5): 480-99. Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
[0073] W000 / 42072, Shields RL, et al. J Biol Chem.
[0074] 2001 Mar 2; 276 (9): 6591-604. Lazar, G. A., et al. Proc Natl Acad Sci USA. 103 (11): 4005-10. W02004 / 029207 WO02004 / 074455 WO99 / 51642 WO2004074455A2 Idusogie EE et al. J Immunol. 2001; 166: 2571-5. Dall'Acqua et al. J Immunol 2006; 177: 1129-1138.
[0075] Moore GL. Et al. nnAbs 2: 2, 181-189; March / April, 2010. Verhoeyn et al. BioEssays, 8:74, 1988. Verhoeyen et al. Science, 239: 1534-1536, 1988. Jones et al. Nature, 321: 522-525, 1986. Riechmann et al. Nature, 332: 323-327, 1988.
[0076] Almagro et al. Frontiers in Bioscience 13, 1619-1633, January 1, 2008. Jakobovits et al., Proc. Natl. Acad. Sci. USA. 90: 2551 (1993). Jakobovits et al., Nature, 362: 255-258 (1993). Bruggermann et al., Year in Immuno., 7:33 (1993). Duchosal et al. Nature 355: 258 (1992) U55,591,669 US 5,598,369 US 5,545,806 US 5,545,807 US 6,150,584 Hoogenboom et al., J. Mol. Biol., 227: 381 (1991).
[0077] Marks et al., J. Mol. Biol. 222: 581-5997 (1991). Vaughan et al. Nature Biotech 14: 309 (1996). W02012 / 041768 Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994).
[0078] 40 Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993).
[0079] 3025515 42 Ryan et al., 1997 Science; 278: 873-876. Cibelli et al., 1998 Science, 280: 1256-1258. W00026357A2 W09004036A1 5 W09517085A1 W00126455A1 W02004050847A2 W02005033281A2 W02007048077A2 W00126455A1 W02007106078A2 Stoger E, et al. Molecular Breeding 9: 149-158, 2002. Fisher R, et al. Vaccine 21 (2003) 820-825. Forthal et al., J Immunol 2010; 185; 6876-6882.
[0080] My JK, et al. Nat Rev Genet.
[0081] 2003 Oct; 4 (10): 794-805. Schillberg S, et al. Vaccine 23 (2005) 1764-1769. U56,013,763 U55,084,559 U55,260,373 U56,399,750 U57,709,209 W02012 / 083425 Ldfdahl S, Guss B, Uhlen M, Philipson L, Lindberg M. Gene for Staphylococcal Protein A. Proc Natl Acad Sci U S A.
[0082] 1983 Feb; 80 (3): 697-701.
[0083] Uhlen M, Guss B, Nilsson B, Gatenbeck S, Philipson L, Lindberg Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J Biol Chem.
[0084] 1984 Feb 10; 259 (3): 1695-702.

权利要求:
Claims (11)
[0001]
REVENDICATIONS1. A method of purifying a monoclonal antibody or a fusion protein between the Fe fragment of an antibody and a second polypeptide, comprising: a) a step of affinity chromatography on a resin having a matrix of a polymer gel of crosslinked methacrylate, on which is grafted protein A, b) a viral inactivation step, c) a cation exchange chromatography step on a resin having a crosslinked agarose gel matrix on which are grafted sulfonate groups (-503-) via dextran spacer arms, d) an anion exchange chromatography step on a hydrophilic polyethersulfone membrane coated with a crosslinked polymer on which quaternary amine groups are grafted (Q) and e) a nanofiltration step with a filter having a double polyethersulfone membrane having a porosity of about 20 nm.
[0002]
2. Method according to claim 1, characterized in that the crosslinked methacrylate polymer gel on which is grafted protein A used in step a) is in the form of beads having a mean diameter of between 30 and 60 [ Inn, advantageously between 40 and 50 [Inn.
[0003]
3. Method according to claim 1 or claim 2, characterized in that the elution buffer used in step a) to elute the antibody is a formate buffer. 25
[0004]
4. Method according to any one of claims 1 to 3, characterized in that the formate buffer used for the elution of the antibody in step a) is used at a molarity of 5 to 10 mM and at a pH between 2.6 and 3.6. 30
[0005]
5. Method according to any one of claims 1 to 4, characterized in that step b) is carried out by incubation for 30 to 120 minutes at a temperature of 20 to 25 ° C in a medium comprising 0.5 to 2 % (v / v) of polyoxyethylene-p-octylphenol (Triton X-100, CAS No. 9002-93-1). 35
[0006]
6. Method according to any one of claims 1 to 5, characterized in that the buffer used in step d) is a trishydroxymethylaminomethane (TRIS) buffer at a concentration of 15 to 25 mM, a pH of 7.5 at 8.5 and a conductivity of 5 to 15 mS / cm. 3025515 44
[0007]
The method according to any one of claims 1 to 6, characterized in that step e) further comprises pre-filtration through a depth filter comprising cellulose fibers, diatomaceous earth and a charged resin. negatively or a polyethersulfone membrane with a porosity of 0.22 [Inn 5 functionalized with SO 3 - groups.
[0008]
8. Method according to any one of claims 1 to 7, characterized in that it further comprises a step of ultrafiltration and / or diafiltration. 10
[0009]
9. Process according to any one of claims 1 to 6, characterized in that it is carried out on a culture supernatant of a clone producing the monoclonal antibody or the fusion protein between the Fe fragment and an antibody and a second polypeptide. 15
[0010]
10. Process according to any one of claims 1 to 7 for the purification of a monoclonal antibody.
[0011]
11. Method according to claim 10, characterized in that the antibody is directed against one of the following antigens: Rhesus D, CD2, CD3, CD4, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD51 ( Integrin alpha-V), CD52, CD80, CTLA-4 (CD152), SLAMF7 (CD319), Her2 / neu, EGFR, EPCAM, CCR4, CEA, FR-alpha, GD2, GD3, HLA-DR, IGF1R (CD221) , phosphatidylserine, TRAIL-R1, TRAIL-R2, Clostridium difficile antigens, Staphylococcus aureus antigens, Cytomegalovirus antigens, Escherichia coli antigens, Respiratory syncytial virus antigens, Hepatitis B virus antigens, Influenza virus antigens A, Pseudomonas aeruginosa serotype IATS 011 antigens, rabies virus antigens, or phosphatidylserine.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2011050071A2|2009-10-20|2011-04-28|Abbott Laboratories|Isolation and purification of anti-il-13 antibodies using protein a affinity chromatography|

---

WO2013009526A1|2011-07-08|2013-01-17|Merck Sharp & Dohme Corp.|Method for purifying fc-fusion protein|

---

WO2013013193A1|2011-07-20|2013-01-24|Zepteon, Incorporated|Polypeptide separation methods|

---

WO2013177115A2|2012-05-21|2013-11-28|Abbvie Inc.|Novel purification of human, humanized, or chimeric antibodies using protein a affinity chromatography|

---

US5260373A|1987-03-13|1993-11-09|Repligen Corporation|Immobilized immunoglobulin-binding proteins|

---

US5084559A|1987-03-27|1992-01-28|Repligen Corporation|Protein a domain mutants|

---

GB8823869D0|1988-10-12|1988-11-16|Medical Res Council|Production of antibodies|

---

US5175384A|1988-12-05|1992-12-29|Genpharm International|Transgenic mice depleted in mature t-cells and methods for making transgenic mice|

---

US6150584A|1990-01-12|2000-11-21|Abgenix, Inc.|Human antibodies derived from immunized xenomice|

---

US5545806A|1990-08-29|1996-08-13|Genpharm International, Inc.|Ransgenic non-human animals for producing heterologous antibodies|

---

US5827690A|1993-12-20|1998-10-27|Genzyme Transgenics Corporatiion|Transgenic production of antibodies in milk|

---

EP0690452A3|1994-06-28|1999-01-07|Advanced Micro Devices, Inc.|Electrically erasable memory and method of erasure|

---

SE9503925D0|1995-11-07|1995-11-07|Pharmacia Biotech Ab|Separation medium for IgG|

---

US6013763A|1996-06-04|2000-01-11|Genentech, Inc.|Peptide variants of protein A|

---

CA2323757C|1998-04-02|2011-08-02|Genentech, Inc.|Antibody variants and fragments thereof|

---

WO2000026357A2|1998-11-02|2000-05-11|Genzyme Transgenic Corp.|Transgenic and cloned mammals|

---

HU0104865A3|1999-01-15|2004-07-28|Genentech Inc|Polypeptide variants with altered effector function|

---

JP2003511048A|1999-10-14|2003-03-25|ジェンジムトランスジェニックスコーポレイション|Method for producing target molecule in transgenic animal and method for purifying the target molecule|

---

US20040002587A1|2002-02-20|2004-01-01|Watkins Jeffry D.|Fc region variants|

---

US7709209B2|2002-03-25|2010-05-04|Ge Healthcare Bio-Sciences Ab|Protein ligands|

---

AT541857T|2002-09-27|2012-02-15|Xencor Inc|OPTIMIZED FC VARIANTS AND MANUFACTURING METHOD THEREFOR|

---

CN1729298A|2002-11-27|2006-02-01|Gtc生物治疗学公司|Modified antibodies stably produced in milk and methods of producing same|

---

JP2006524039A|2003-01-09|2006-10-26|マクロジェニクス，インコーポレーテッド|Identification and production of antibody containing mutant Fc region and use thereof|

---

AR047720A1|2003-09-30|2006-02-15|Bio Sidus S A|A PROCESS FOR THE OBTAINING OF TRANSGENIC MAMMALS THAT PRODUCE MILK PROTEINS AND MAMMALS PRODUCED BY THIS PROCESS|

---

PL1945666T3|2005-10-21|2013-09-30|Genzyme Corp|Antibodies with enhanced antibody-dependent cellular cytoxicity activity, methods of their production and use|

---

US7531632B2|2006-02-16|2009-05-12|Gtc Biotherapeutics, Inc.|Clarification of transgenic milk using depth filtration|

---

US20120258496A1|2010-09-27|2012-10-11|Boehringer Ingelheim International Gmbh|Production of low fucose antibodies in h4-ii-e rat cells|

---

US9051375B2|2010-12-21|2015-06-09|The University Of Western Ontario|Alkali-resistant variants of protein A and their use in affinity chromatography|US20180072769A1|2015-03-23|2018-03-15|Alexion Pharmaceuticals, Inc.|Virus filtration|

---

US11214789B2|2016-05-03|2022-01-04|Flodesign Sonics, Inc.|Concentration and washing of particles with acoustics|

---

BR112020009259A2|2017-11-14|2020-10-20|Bio-Sourcing S.A.|antibody purification|

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FR3076294B1|2017-12-29|2022-01-28|Lab Francais Du Fractionnement|METHOD FOR PURIFYING ANTIBODIES FROM RAW MILK|

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CN110128537A|2018-02-09|2019-08-16|鲁南制药集团股份有限公司|A kind of purification process of aglycosylated anti-PD-1 monoclonal antibody|

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EP3643322A1|2018-10-26|2020-04-29|Mabion SA|Low aggregate anti cd20 ligand formulation|

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WO2020263717A1|2019-06-26|2020-12-30|Merck Sharp & Dohme Corp.|Process for separating antigen-binding polypeptide monomers comprising one or more immunoglobulin single variable domains from aggregates|

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优先权:

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申请号 | 申请日 | 专利标题

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FR1458346A|FR3025515B1|2014-09-05|2014-09-05|PROCESS FOR PURIFYING MONOCLONAL ANTIBODY|FR1458346A| FR3025515B1|2014-09-05|2014-09-05|PROCESS FOR PURIFYING MONOCLONAL ANTIBODY|

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EP15759793.1A| EP3189075B1|2014-09-05|2015-09-04|Method for purification of monoclonal antibodies|

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US15/508,577| US10363496B2|2014-09-05|2015-09-04|Method for purification of monoclonal antibodies|

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CA2959947A| CA2959947A1|2014-09-05|2015-09-04|Method for purification of monoclonal antibodies|

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PCT/EP2015/070298| WO2016034726A1|2014-09-05|2015-09-04|Method for purification of monoclonal antibodies|

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TR2019/03667T| TR201903667T4|2014-09-05|2015-09-04|PROCESS FOR PURIFICATION OF MONOCLONAL ANTIBODIES.|

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ES15759793T| ES2720255T3|2014-09-05|2015-09-04|Purification procedure of a monoclonal antibody|